bioRxiv preprint doi: https://doi.org/10.1101/852939; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Patel, A et al. 1 Integrative genomic and epigenomic analyses identify a distinct role of c-Myc and L-Myc for lineage determination in small cell lung cancer Ayushi S. Patel1,2, Seungyeul Yoo3,4, Ranran Kong1,2,5, Takashi Sato1,2, Maya Fridrikh1,2, Abhilasha Sinha1,2, German Nudelman6, Charles A. Powell1,2, Mary Beth Beasley7, Jun Zhu2,3,4, Hideo Watanabe1,2,3,8* 1Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 2Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 4Sema4, a Mount Sinai venture, Stamford, CT 06902, USA. 5Department of Thoracic Surgery, The Second Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, Shaanxi, 710004, China 6 Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 7Department of Pathology and Laboratory Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 8Lead Contact *Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/852939; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Patel, A et al. 2 Abstract Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer with a dismal prognosis. The standard-of-care remains to be uniform treatment with chemotherapy and radiotherapy, even though emerging evidence suggests disease heterogeneity. In primary SCLC, the gene loci for Myc family members are amplified mutually exclusively, their expression is correlated with unique neuroendocrine markers and distinct histopathology of xenografts from SCLC cell lines and murine SCLC. In this study, we explore a novel role of c-Myc and L-Myc as lineage specific factors to bridge the gap between SCLC molecular subtypes and histological classification. Integrated analyses of a Bayesian network generated from primary tumor mRNA expression and chromatin state profiling of cell lines showed that Myc family members impart distinct transcriptional programs associated with lineage state; wherein L-Myc was enriched for neuronal pathways and c-Myc for Notch signaling and epithelial-to-mesenchymal transition. Genetically engineering the exchange of c-Myc with L-Myc in c-Myc amplified SCLC revealed the insufficiency of L-Myc to induce lineage switch but the requirement for c-Myc to maintain NeuroD1-lineage state. In contrast, exogenous expression of c-Myc in L-Myc amplified ASCL1- SCLC revealed incompatibility with c-Myc expression in those SCLC, that eventually trans- differentiated to NeuroD1-SCLC accompanied by variant histopathological features. Transcriptomic profiling of trans-differentiated cells revealed Notch signaling target, Re-1 silencing transcription factor (REST) was induced with c-Myc expression mediating the alteration of ASCL1 expression. Collectively, our findings reveal a previously undescribed role for historically defined general oncogenes, c-Myc and L-Myc, in regulating lineage plasticity across molecular subtypes as well as histological classes. bioRxiv preprint doi: https://doi.org/10.1101/852939; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Patel, A et al. 3 Introduction Small cell lung cancer (SCLC) represents about 15% of all lung cancers with a median survival time of approximately only 10 months and 5-year overall survival at 6% (Siegel et al., 2019). SCLC is characterized by neuroendocrine differentiation, rapid growth, early metastatic spread and poor prognosis (Gazdar et al., 2017). The standard of care has remained to be cytotoxic chemotherapies for decades, mainly etoposide and a platinum agent (Alvarado-Luna et al., 2016), that is only temporarily effective for the vast majority of patients (Pietanza et al., 2015). Even with recent developments in immune checkpoint inhibitors, activity against SCLCs when combined with chemotherapy has been marginal with a modest improvement on the median survival (10.3 months vs. 12.3 months) for extensive stage SCLCs (Horn et al., 2018). These data reflect the urgent need for effective therapeutics for patients with SCLC. The lack of effective therapeutics for SCLC stands in stark contrast to the breadth of targeted therapies for non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma (Rudin et al., 2019). This progress in drug development for NSCLCs is largely attributable to a more comprehensive understanding of molecular subtypes and identifying targetable driver oncogenes (Zappa et al., 2016). Therefore, better characterization of the molecular subtypes of SCLC should aid future drug development and patient stratification for targeted therapies. Morphological classification of SCLC was noted over three decades ago when human SCLC cell lines were implanted as xenografts and distinguished as two primary subtypes: classical SCLC and variant SCLC (Carney et al., 1985; Gazdar et al. 1985). The classical subtype features relatively small cells with high nuclear:cytoplasm ratio while the variant subtype exhibits relatively larger cells and moderate amounts of cytoplasm. The variant subtype is correlated with reduced expression of neuroendocrine markers and frequent MYC bioRxiv preprint doi: https://doi.org/10.1101/852939; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Patel, A et al. 4 amplification (Gazdar et al. 1985). The variant features were later identified in primary specimens (Beredensen et al., 1988). However, the World Health Organization (WHO) classification, updated in 2015, recognizes SCLC as a homogenous disease with neuroendocrine features defined by histological features including small cells, scant cytoplasm, nuclear morphology with fine granular chromatin and lacking prominent nucleoli, reminiscent of the features of classical SCLC with no further sub-classifications (Travis et al., 2015). The variant histopathology observed in xenografts of SCLC cell lines, according to the WHO classification, would likely be recognized as “combined SCLC” that includes a component of either large-cell neuroendocrine carcinomas (LCNEC) or NSCLC, potentially reflective of the discrepancies between clinical practice and experimental findings (Travis et al., 2015, Rekhtman, 2010). More recent efforts to distinguish SCLC molecular subtypes include profiling gene expression and genome-wide methylation in primary human tumors and patient derived xenografts (PDX), that revealed three clusters, with a dichotomy between achaete-scute homologue 1 (ASCL1) and neurogenic differentiation factor 1 (NeuroD1) expression, in addition to a cluster with low expression of both (Poirier et al., 2015). The expression of ASCL1 and NeuroD1 has been implicated to confer SCLC heterogeneity by imparting distinct transcriptional profiles (Borromeo et al., 2016). The third neuroendocrine-low cluster led to the further classification into two subtypes characterized by transcriptional driver YAP1 or POU class 2 homeobox 3 (POU2F3) (McColl et. Al., 2017, Huang et al., 2018). SCLC cell line xenograft histology has been correlated with these contrasting factors where variant SCLC was positively correlated with a higher NeuroD1:ASCL1 ratio and classical SCLC was positively correlated with the inverse in SCLC cell lines (Poirier et al., 2013). A bioRxiv preprint doi: https://doi.org/10.1101/852939; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Patel, A et al. 5 recent study showed that additional c-Myc overexpression, in a classical SCLC genetically engineered mouse model (GEMM) with conditional loss of Rb1 and Trp53 mouse, can drive the progression of murine SCLC to variant histopathology with reduced neuroendocrine gene expression including ASCL1 but displayed higher NeuroD1 (Mollaoglu et al., 2017). On the contrary, the classical SCLC GEMM harbored stochastic MYCL amplifications or overexpression associated with classical SCLC histopathology (Meuwiseen et al., 2003). Recapitulating Myc family members in SCLC GEMMs has provided insight in the role for tumorigenesis and contribution to histopathological characteristics (Brägelmann et al., 2017, Kim et al., 2016). c-Myc (MYC) and L-Myc (MYCL) belong to the MYC family of basic helix-loop-helix (bHLH) transcription factors. The paralogs contain functionally relevant highly conserved amino acid sequences and are structurally homologous (DePinho et al., 1987b, Conacci-Sorell et al., 2014). c-Myc is a well-characterized oncogene; L-Myc although understudied is implicated to have a similar oncogenic role. Amplification of Myc family members are mutually exclusive and overall account for ~20% of SCLC and overexpression for